Lubomir S. Hnilica

Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

Abstract A method of rapidly identifying lectin-binding glycoproteins separated by polyacrylamide gel electrophoresis is described. The method is particularly useful for comparing the glycoprotein content of different cell types and fractions. Normal rat liver, Novikoff hepatoma, and rat mammary tumor cell line 13762 MAT-B were fractionated to give purified nuclei and other fractions defined by their sedimentation properties in low ionic strength buffer. The subcellular fractions were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, transferred to nitrocellulose sheets, and localized by an immunochemical method to identify lectin-binding activities. The localization pattern of concanavalin A and wheat germ agglutinin-binding activities in the fractions from the three cell types showed the greatest similarities between the glycoprotein contents of normal liver and Novikoff hepatoma fractions. On a per-cell basis the purified nuclei from each of the cell types contained less activity overall than did other particulate cell fractions. Washing the nuclei from normal liver and Novikoff hepatoma, but not MAT-B cells, in nonionic detergent removed or depressed most of the lectin-binding activities. However, two major bands were unaffected by the detergent. One of these localized with wheat germ agglutinin at an apparent molecular weight of 62,000 in the nuclei of all three cell types. The other localized with concanavalin A at an apparent molecular weight of 200,000 in normal liver and Novikoff hepatoma nuclei.

Nuclear protein transitions in rat testis spermatids.

Abstract Dramatic transitions occur in the nuclear proteins during spermiogenesis in rats. In order to determine more precisely when these transitions occur, we have employed centrifugal elutriation, velocity sedimentation at unit gravity, centrifugation in metrizamide gradients, and sonication to obtain relatively homogeneous populations of testis cells and nuclei. The results indicate that histones are present in step 1–8 spermatid nuclei but are not detectable after step 12. Nuclear proteins designated TP and TP2 are not detectable in step 1–8 spermatids but are present and actively synthesized in step 13–15 spermatids. These two proteins are turned over within 5 days after synthesis. A spermatidal basic nuclear protein, designated TP3, and the sperm basic nuclear protein, S1, are present in step 16–19 spermatids. Biochemical characterization of TP2 and TP3 are presented.

Gamma-Radiation-Induced Crosslinking of Cell-Specific Chromosomal Nonhistone Protein-DNA Complexes in HeLa Chromatin

Specific antisera were obtained by injecting rabbits or goats with dehistonized HeLa cell chromatin. Gamma irradiation (10 to 100 krad) of the isolated chromatin increased its immunological reactivity with these specific antisera. Conversely, irradiation of the isolated chromosomal nonhistone proteins or DNA followed by reconstitution resulted in a nearly complete loss of immunological reactivity of the reconstituted complexes. Selective protein dissociation experiments indicated that radiation crosslinked the active nonhistone proteins to the DNA. This interpretation was further substantiated by polyacrylamide gel electrophoresis of the crosslinked proteins.

DNA-protein crosslinking by heavy metals in Novikoff hepatoma☆

Crosslinking of proteins to DNA was studied in live intact Novikoff ascites hepatoma cells exposed in vitro to salts of chromium VI, III, and II, nickel II, cadmium II, and to CoCl2, As2O3, and AlK(SO4)2. DNA-protein complexes were separated by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and assayed by polyacrylamide gel electrophoresis. Hexavalent chromium compounds formed DNA-protein complexes very efficiently. The trivalent, poorly soluble, cupric chromite was nearly as efficient crosslinker as hexavalent Cr, perhaps because phagocytosis facilitated its entry into the cells. The more basic divalent form produced hardly any crosslinks. Most of the crosslinked proteins were common to all of the chromium salts employed. Nickel salts formed DNA-protein crosslinks less efficiently. Most proteins crosslinked by this metal had a high molecular weight ranging from 94,000 to 200,000. There was little qualitative difference between the crosslinked protein patterns for several various nickel (II) salts. Similar results were obtained for cells incubated with cadmium salts. Most of the proteins crosslinked by cadmium had high molecular weights and were similar to those crosslinked by nickel (II). Relatively weak, but significant, crosslinking was also observed when the Novikoff hepatoma cells were exposed to CoCl2, As2O3, or AlK(SO4)2.

Nonhistone Protein Antigens

Assuming that the genetic information of the eukaryotic cell is contained within the nuclear DNA, differentiation can be viewed as transcriptional restriction of DNA, specific for each differentiated cell type. Consequently, detailed knowledge of biological and biochemical properties of nuclear proteins and their functional associations with DNA is essential for our understanding of mechanisms governing cellular proliferation and differentiation. Since immunological methods are perhaps the most powerful tools available for probing the heterogeneity, diversity, and specificity of macromolecular components, it can be anticipated that their application to the research on chromosomal proteins will contribute substantially to our knowledge of chromatin biology and biochemistry.

Lactoferrin: nuclear localization in the human neutrophilic granulocyte?

Conditions were established whereby nuclear or cytoplasmic immunocytochemical localization of lactoferrin was observed in the human peripheral blood granulocyte. A positive nuclear staining reaction was obtained when cells were either not fixed or treated with most fixatives. However, treatment of blood smears with formaldehydeacetone prior to the immunocytochemical localization gave a cytoplasmic staining reaction. A method was developed that allowed us to examine proteins obtained from granulocyte nuclei isolated from fixed cells. Only a trace amount of lactoferrin was detected in the electrophoretically separated nuclear proteins obtained from granulocytes treated for 1 min with formaldehyde-acetone. However, lactoferrin was major protein component in nuclei isolated from untreated cells, cells treated with other fixatives, or cells preincubated in buffered saline prior to formaldehyde-acetone fixation. formaldehyde-acetone treatment of nuclear materials containing lactoferrin did not extract lactoferrin or mask it from detection, thus indicating that lactoferrin found in the mature neutrophilic granulocyte nucleus is most likely acquired from other cellular organelles during tissue processing.

Crosslinking of chromosomal proteins to DNA in HeLa cells by UV, gamma radiation and some antitumor drugs

Immunochemical techniques were used to investigate the protein-DNA crosslinking by ultraviolet (UV) and gamma radiation as well as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or cis- and trans-diamminedichloroplatinum II (cis-DDP and trans-DDP). Antisera to 0.35 M NaCl extract and 0.35 M NaCl residue of HeLa nuclei were employed. Both gamma and UV irradiation, exposure to cis- or trans-DDP and, to a lesser extent, BCNU, resulted in crosslinking of various antigens to the DNA. Although several antigens were crosslinked by all the employed agents, other exhibited agent-specific crosslinking patterns.

Cell specific antiserum to chromosome scaffold proteins.

Antiserum has been raised to a chromosomal protein fraction specific for Hela cells. The immunoactivity is located in the transcriptionally inactive regions of log phase chromatin. Digestion of metaphase chromosomes results in the purification of the immunoactivity in the scaffold region of the chromosomes. Extensive nuclease digestion of the scaffolds results in loss of activity. The data suggest that some of the proteins in the scaffold area are both tight binding and cell specific and may therefore play a sophisticated role in gene expression.

Modified method of silver staining of proteins in polyacrylamide gels.

The very sensitive and reliable silver staining method to visualize proteins in polyacrylamide gels described by Wray et al. (Anal. Biochem. (1981) 118, 197-203) fails when the protein sample contains nucleic acids and/or metals. By washing the polyacrylamide gels in acetic acid and repeatedly in methanol immediately following electrophoresis and then using the procedure of Wray et al., many gels otherwise unstainable may be stained with a high degree of reliability. This method allows visualization of a minute amount of proteins in samples containing high amounts of DNA and metals.

Chapter 17 Methods for Selective Extraction of Chromosomal Nonhistone Proteins

Publisher Summary This chapter describes the methods for selective extraction of chromosomal nonhistone proteins (NP). During rapid cell proliferation, chromosome puffing, and chromosomal stimulation, there is an increase in the content, synthesis, and metabolic activity of chromosomal nonhistone proteins. Presently, there are no methods available for a complete separation of all biologically active proteins by a single standard procedure. Techniques taking advantage of particular biological properties of chromosomal nonhistone proteins, such as enzymic activity, transcriptional regulations, and immunological specificity, are most useful. The chapter describes the fractionation scheme, which is based on solubility of the three protein groups in salt and urea at various pH values. Although fraction NP binds selectively to homologous DNA, its separation from histones in sodium succinate buffer is facilitated by its insolubility at relatively low pH. The NP fraction is heterogeneous. Isolated chromatin can be fractionated by a variety of techniques into transcriptionally active and inactive fractions. If the fractionation of chromatin is accomplished by shearing and subsequent sucrose density gradient centrifugation or divalent cation precipitation, the immunologically tissue-specific proteins are selectively accumulated in the extended, transcriptionally active chromatin.